Nuclear receptors (NRs) are transcriptional regulators of metabolism. A major goal of this laboratory is to understand the biological importance of transcriptional repression by unliganded NRs. The orphan receptor Rev-erba has been particularly enlightening because it lacks an activation domain and is a constitutive represser. Transcriptional repression by Rev-erba is a key molecular component of the negative limb of the cell autonomous circadian clock. Normal circadian rhythms are disrupted both by absence of Rev-erba, and by unregulated persistence of its function. In the prior period, we showed that Rev-erba recruitment of the N-CoR/HDAC3 corepressor complex is critical for repression of circadian genes, and that modulation of Rev- erba protein stability initiates and synchronizes cellular clocks. Here we will determine the cellular mechanisms regulating Rev-erba activity, and explore the role of Rev-erba in regulating circadian activity and metabolic functions in liver and adipocytes. Specific Aim 1 is to determine molecular mechanisms regulating Rev-erba activity. Rev-erba protein is stabilized by GSKSp-dependent phosphorylation, and we hypothesize that this modification regulates association with a specific E3 ligase. Biochemical and molecular approaches will be taken to identify and validate the role of specific components of this complex, and determine their involvement in normal clock function. Preliminary data indicate that Rev-erba activity is also regulated by heme binding. We will test the hypothesis that this plays a critical role in regulating the normal role of Rev-erba in the cell autonomous clock. Specific Aim 2 is to determine the role of mechanisms regulating Rev-erba activity on circadian and metabolic physiology. We hypothesize that the mechanisms regulating cellular Rev-erba activity have important effects on circadian and metabolic processes. This will be tested by expressing informative Rev-erba mutants in liver, where Rev-erba normally regulates circadian gene expression and metabolism. Specific Aim 3 is to understand the role of Rev- erba in adipocyte biology. Rev-erba is induced during adipogenesis, and we hypothesize that it regulates not only adipocyte differentiation, but also the physiological functions of the mature adipocyte, including adipokine production and lipolysis, both of which display circadian rhythms in vivo. We will explore the role of Rev-erba activity in the basal and circadian regulation of these adipocyte genes as well as adipocyte metabolic functions. Insights gained from this work will shed new light on the transcriptional and post- transcriptional control of circadian and metabolic physiology, as well as interrelationships between circadian rhythms and metabolism. This has the potential to lead to new understanding of circadian and metabolic disorders, including obesity, diabetes, and cardiovascular disease.